Chloride ion is the major extra-cellular anion. Thus, it is significantly involved in maintaining proper hydration, osmotic pressure, and normal anion-cation balance in warm-blooded animals like humans. Clinically, chloride ion loss is associated with chronic pyelonephritis. Low serum chloride values may be observed in those types of metabolic acidosis (e.g., diabetic acidosis and renal failure) that are caused by excessive production or diminished secretion of acids. High serum chloride values are observed in dehydration and in conditions causing decreased renal blood flow, such as congestive heat failure. Accordingly, an accurate, economical and convenient method for determining chloride ion levels in body fluids is a very valuable tool for the clinical chemist.
A variety of methods have been used to measure chloride ions in body fluids such as serum. Among the most common of these methods are coulometric-amperometric titrations, end point titrations using mercuric nitrate and diphenylcarbazone, and colorimetric methods using mercuric thiocyanate-ferric nitrate. (Tietz, N. W. ed., Fundamentals of Clinical Chemistry, 2nd Ed., pp 879-884, 1976, WB Saunders Co., Philadelphia, PA).
Due to the complexity of many body fluids, e.g., serum, it is usually necessary with most of the prior art methods, except for the colorimetric-amperometric method, to remove protein from the sample before making chloride measurements. This is usually accomplished by making a protein-free filtrate using protein precipitating acids (e.g. tungstic acid) or through dialysis, where the dialyzing membrane retains large molecules, such as serum proteins, and allows only small ions such as chloride to pass through.
The drawback to the aforementioned procedures, however, is that removal of protein from the sample is not well suited for most clinical chemistry analyzers or manual assays. The precipitation methods require additional reagents, mixing, centrifugation or filtration, and decanting prior to the actual chloride measurement. With the dialysis procedures, specially designed equipment is necessary and procedures using this methodology have largely been limited to Auto Analyzer.RTM. methods. In the case of serum chloride, measurements made without protein removal on a centrifugal or similar clinical analyzer as for example, a COBAS BIO.RTM. Centrifugal Analyzer (Roche Diagnostic Systems, Division of Hoffmann-La Roche Inc., Nutley, N.J. 07110) using the mercuric thiocyanate-ferric nitrate method may result in errors being made if sample blanks are ignored.
Frequently, the sample of body fluid being assayed for chloride ion content will contain endogeneous compounds, e.g., lipids or chylomicrons (lipids bound to protein) that absorb or scatter light at the wavelength maximum of the ferric thiocyanate complex formed when chloride ion concentration is measured using the ferric nitrate-mercuric thiocyanate procedure. Thus, certain samples, especially lipemic sera, may cause over-recovery of the chloride ion concentration in the sample. This can be illustrated by Equation 1, where A.sub.1 +A.sub.2 +. . . A.sub.n are compounds present in the sample that absorb at or near the wavelength of the ferric thiocyanate complex being measured.